Abstract The objective of the proposed studies is to develop and characterize animal model for studying the progression of estrogen-dependent cancer to estrogen independent state. The need for developing such animal model is obvious because practically all invasive human breast cancers, although they may initially respond to hormonal therapy, will progress to hormone independent state, and there is no suitable and well-characterized animal model available to study this process in details. A few years ago, it was shown that although over 90% of N-methyl-N-nitrosourea (MNU)-induced rat mammary cancers either become impalpable or show more than 50% reduction in size after ovariectomy, a high percentage of these tumors show re-growth with prolonged time after ovariectomy. This animal model will be used in this proposal for further development and characterization. It is hypothesized that mammary cancers that show a renewed growth after ovariectomy initially continue to depend on estrogen for growth by developing an increased sensitivity to estrogen and by enhancing local synthesis of estrogen after estrogen deprivation, but gradually lose their need for estrogen to acquire complete estrogen independence. The following two specific aims are proposed: 1. To investigate what changes are seen in gene expression when mammary cancers progress from a state of ovarian-dependent growth to re-growth in the absence of ovarian hormones. Here, microarray analysis will be used to determine global changes in gene expression of mammary tumors when they progress from hormone-dependent to hormone-independent state. 2. To investigate the importance of estrogen/estrogen receptor activity in mammary cancers of ovariectomized animals. In this specific aim, it will be investigated if the estrogen receptor continues to be involved in growth regulation of mammary cancers that grow after ovariectomy of the tumor-bearing animals. Public Health Statement Breast cancer is one of the major causes of cancer-related deaths of women worldwide. It is estimated that approximately 200,000 women were diagnosed with invasive breast cancer and over 40,000 died of the disease in the USA in 2005. Much effort has been spent on elucidating the etiology of breast cancer, but our understanding of what causes this disease is still fragmental. It has been difficult to demonstrate that a single factor is the main cause for breast cancer development, but rather that a complex of genetic and environmental factors play a role. Approximately 70% of invasive breast cancers in women at diagnosis are estrogen receptor (ER)-positive and up to 70% of these tumors, or 50% of all diagnosed invasive breast cancers, regress upon hormone (antiestrogen, aromatase inhibitor) therapy. However, although the ER-positive tumors respond initially well to hormone therapy, the benefits are not permanent and relapses inevitably occur usually within months or a few years of treatment, giving rise to cancers that appear to grow completely independent of estrogen stimulation. Therefore, it is obvious that estrogen/ER are fundamental growth regulators at relatively early states of approximately 50% of all breast cancers. Much effort has been spent on trying to understand fully how breast cancers that initially depend on hormone for growth develop complete hormonal independence. One of the problems hampering progress in understanding this phenomenon is an appropriate animal model. In this application, we propose to develop and characterize a chemically-induced rat mammary cancer model to study the progression of hormone-dependent breast cancer to hormone-independent state. Preliminary studies presented in this application show remarkable similarities between this animal model and human breast cancer. However, the mechanism by which breast cancers acquire independence from hormonal stimulation for their growth is still being defined. We propose here to monitor the changes that occur in breast cancers as they progress from estrogen dependence to complete independence from estrogen for growth, with the ultimate goal of determining how these differences in growth regulation may be utilized to develop a more robust treatment against breast cancer, particularly after it has progressed to estrogen independent state.